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Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

Rodríguez-Esteban G, González-Sastre A, Rojo-Laguna JI, Saló E, Abril JF - BMC Genomics (2015)

Bottom Line: These results are accessible via web for the community of researchers.DGE is a valuable tool for gene discovery, quantification and annotation.The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona (UB), and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Av. Diagonal 643, Barcelona, 08028, Catalonia, Spain. gresteban@scientist.com.

ABSTRACT

Background: The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking.

Results: Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC.

Conclusions: DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

No MeSH data available.


Related in: MedlinePlus

Smed-nf-Y gene complex is required for the proper neoblast differentiation and localization.A - WISH shows that the three Smed-nf-Y genes are expressed ubiquitously and in the cephalic ganglia, and one day after irradiation their expressions decrease. B - Double FISH of Smed-nf-YA, Smed-nf-YB-2, and Smed-nf-YC together with the neoblast marker Smed-h2b shows colocalization with the NF-Y subunits (arrowheads), demonstrating the expression of this complex in neoblasts as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 12A to check each channel of fluorescence separately. C - Smed-nf-Y(RNAi) animals regenerate thinner blastemas with non well formed eyes and shape defects, and fail to differentiate a proper brain, with reduced cephalic ganglia as revealed with Smed-gpas. FISH with the neoblast marker Smed-h2b shows an accumulation of neoblasts in the region in front of the eyes while the early progeny marker Smed-nb.21.11e reveals a decrease of early postmitotic cells in Smed-nf-Y(RNAi) animals. D - Immunohistochemistry with the mitotic marker α-H3P shows a reduction in the number of mitosis. E - Quantification with category markers indicate a significant increase of Smed-h2b+ cells in Smed-nf-YB-2(RNAi) and Smed-nf-YC(RNAi) animals and a significant decrease of nb.21.11e+ cells in all of the RNAi animals, whereas Smed-agat-1+ cells do not show significant changes (p < 0.001, t-test). Counts are referred to the whole body. ph: pharynx. All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after one round of injection.
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Fig7: Smed-nf-Y gene complex is required for the proper neoblast differentiation and localization.A - WISH shows that the three Smed-nf-Y genes are expressed ubiquitously and in the cephalic ganglia, and one day after irradiation their expressions decrease. B - Double FISH of Smed-nf-YA, Smed-nf-YB-2, and Smed-nf-YC together with the neoblast marker Smed-h2b shows colocalization with the NF-Y subunits (arrowheads), demonstrating the expression of this complex in neoblasts as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 12A to check each channel of fluorescence separately. C - Smed-nf-Y(RNAi) animals regenerate thinner blastemas with non well formed eyes and shape defects, and fail to differentiate a proper brain, with reduced cephalic ganglia as revealed with Smed-gpas. FISH with the neoblast marker Smed-h2b shows an accumulation of neoblasts in the region in front of the eyes while the early progeny marker Smed-nb.21.11e reveals a decrease of early postmitotic cells in Smed-nf-Y(RNAi) animals. D - Immunohistochemistry with the mitotic marker α-H3P shows a reduction in the number of mitosis. E - Quantification with category markers indicate a significant increase of Smed-h2b+ cells in Smed-nf-YB-2(RNAi) and Smed-nf-YC(RNAi) animals and a significant decrease of nb.21.11e+ cells in all of the RNAi animals, whereas Smed-agat-1+ cells do not show significant changes (p < 0.001, t-test). Counts are referred to the whole body. ph: pharynx. All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after one round of injection.

Mentions: In the sexual strain of S. mediterranea, an NF-YB is necessary to maintain spermatogonial stem cells [91]. We have isolated a different NF-YB subunit (NF-YB-2), and also a member of the other two subunits (NF-YA and NF-YC). WISH shows that the three genes are expressed ubiquitously and in the cephalic ganglia (Figure 7A). Moreover, the expression decrease one day after irradiation indicating a linkage with stem cells, as described in other organisms [92]. Double FISH of each NF-Y subunit together with Smed-h2b confirms the expression of this complex in neoblasts and also in some determined cells (Figure 7B and Additional file 12A).Figure 7


Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

Rodríguez-Esteban G, González-Sastre A, Rojo-Laguna JI, Saló E, Abril JF - BMC Genomics (2015)

Smed-nf-Y gene complex is required for the proper neoblast differentiation and localization.A - WISH shows that the three Smed-nf-Y genes are expressed ubiquitously and in the cephalic ganglia, and one day after irradiation their expressions decrease. B - Double FISH of Smed-nf-YA, Smed-nf-YB-2, and Smed-nf-YC together with the neoblast marker Smed-h2b shows colocalization with the NF-Y subunits (arrowheads), demonstrating the expression of this complex in neoblasts as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 12A to check each channel of fluorescence separately. C - Smed-nf-Y(RNAi) animals regenerate thinner blastemas with non well formed eyes and shape defects, and fail to differentiate a proper brain, with reduced cephalic ganglia as revealed with Smed-gpas. FISH with the neoblast marker Smed-h2b shows an accumulation of neoblasts in the region in front of the eyes while the early progeny marker Smed-nb.21.11e reveals a decrease of early postmitotic cells in Smed-nf-Y(RNAi) animals. D - Immunohistochemistry with the mitotic marker α-H3P shows a reduction in the number of mitosis. E - Quantification with category markers indicate a significant increase of Smed-h2b+ cells in Smed-nf-YB-2(RNAi) and Smed-nf-YC(RNAi) animals and a significant decrease of nb.21.11e+ cells in all of the RNAi animals, whereas Smed-agat-1+ cells do not show significant changes (p < 0.001, t-test). Counts are referred to the whole body. ph: pharynx. All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after one round of injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494696&req=5

Fig7: Smed-nf-Y gene complex is required for the proper neoblast differentiation and localization.A - WISH shows that the three Smed-nf-Y genes are expressed ubiquitously and in the cephalic ganglia, and one day after irradiation their expressions decrease. B - Double FISH of Smed-nf-YA, Smed-nf-YB-2, and Smed-nf-YC together with the neoblast marker Smed-h2b shows colocalization with the NF-Y subunits (arrowheads), demonstrating the expression of this complex in neoblasts as well as in differentiated cells (asterisk). DAPI labels the cell nuclei. See Additional file 12A to check each channel of fluorescence separately. C - Smed-nf-Y(RNAi) animals regenerate thinner blastemas with non well formed eyes and shape defects, and fail to differentiate a proper brain, with reduced cephalic ganglia as revealed with Smed-gpas. FISH with the neoblast marker Smed-h2b shows an accumulation of neoblasts in the region in front of the eyes while the early progeny marker Smed-nb.21.11e reveals a decrease of early postmitotic cells in Smed-nf-Y(RNAi) animals. D - Immunohistochemistry with the mitotic marker α-H3P shows a reduction in the number of mitosis. E - Quantification with category markers indicate a significant increase of Smed-h2b+ cells in Smed-nf-YB-2(RNAi) and Smed-nf-YC(RNAi) animals and a significant decrease of nb.21.11e+ cells in all of the RNAi animals, whereas Smed-agat-1+ cells do not show significant changes (p < 0.001, t-test). Counts are referred to the whole body. ph: pharynx. All the experiments are done on bipolar regenerating trunks, at 11 days of regeneration after one round of injection.
Mentions: In the sexual strain of S. mediterranea, an NF-YB is necessary to maintain spermatogonial stem cells [91]. We have isolated a different NF-YB subunit (NF-YB-2), and also a member of the other two subunits (NF-YA and NF-YC). WISH shows that the three genes are expressed ubiquitously and in the cephalic ganglia (Figure 7A). Moreover, the expression decrease one day after irradiation indicating a linkage with stem cells, as described in other organisms [92]. Double FISH of each NF-Y subunit together with Smed-h2b confirms the expression of this complex in neoblasts and also in some determined cells (Figure 7B and Additional file 12A).Figure 7

Bottom Line: These results are accessible via web for the community of researchers.DGE is a valuable tool for gene discovery, quantification and annotation.The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona (UB), and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Av. Diagonal 643, Barcelona, 08028, Catalonia, Spain. gresteban@scientist.com.

ABSTRACT

Background: The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking.

Results: Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC.

Conclusions: DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

No MeSH data available.


Related in: MedlinePlus